Tag Archives: withering syndrome

DNA Extraction– Ava Withering Syndrome Transmission Study Tissues

Isolated DNA from 20 tissue samples provided by Ava. Presumably, the tissues were digestive gland and I believe they were preserved in ethanol. The list of samples are listed below.

DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s protocol with the following options:

  • Samples were briefly homogenized (due to their stiffness resulting from ethanol fixation) in the InhibitEX Buffer using disposable plastic pestles.
  • Homogenized tissue was incubated at 95C to maximize cell lysis
  • Followed “human DNA analysis” protocol for remainder of protocol (to maximize sample recovery)
  • Eluted DNA with 100μL Buffer ATE

Samples will be quantified tomorrow.

Samples were stored at 4C in FSH240 in the box “Ava WS Transmission DNA Extractions by Sam Box 1″. See the master spreadsheet at the bottom of this post for specific sample locations within this box.

 

Cage Accession Weight (mg)
Control 15:09-1 116
Control 15:09-2 68
Control 15:09-3 138
Control 15:09-10 135
Control 15:09-11 96
Control 15:09-22 100
Control 15:09-23 117
Control 15:09-32 51
Control 15:09-33 115
Control 15:09-34 190
Control 15:10-30 103
Control 15:10-31 83
Control 15:10-32 116
Control 15:10-65 190
Control 15:10-66 112
Control 15:11-142 89
Control 15:11-143 94
Control 15:11-144 94
Control 15:11-153 206
Control 15:11-154 116

Master spreadsheet for these, and future, samples for this project (Google Sheet): Ava WS Transmission DNA Extractions

 

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DNA Extraction & Quantification – Ava Withering Syndrome Transmission Study Tissues

Isolated DNA from 27 tissue samples provided by Ava. Presumably, the tissues were digestive gland and I believe they were preserved in ethanol. The list of samples can be seen in the results below.

DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s protocol with the following options:

  • Samples were briefly homogenized (due to their stiffness resulting from ethanol fixation) in the InhibitEX Buffer using disposable plastic pestles.
  • Homogenized tissue was incubated at 95C to maximize cell lysis
  • Followed “human DNA analysis” protocol for remainder of protocol (to maximize sample recovery)
  • Eluted DNA with 100μL Buffer ATE

After extraction, the samples were quantified using the Roberts Lab Qubit 3.0 (Life Technologies) using the Qubit ds DNA BR reagents. Used 1μL of each sample.

Samples were stored at 4C in FSH240 in the box “Ava WS Transmission DNA Extractions by Sam Box 1″. See the master spreadsheet at the bottom of this post for specific sample locations within this box.

Results:

cage_number accession_number concentration(ng/μL)
9 15:10-29 92
2 15:10-40 114
2 15:10-41 102
2 15:10-42 96
2 15:10-43 101
2 15:10-44 128
11 15:8-95 73
11 15:8-96 74
11 15:8-97 73
11 15:8-98 130
11 15:8-99 42
1 15:8-100 106
1 15:8-101 96
1 15:8-102 91
1 15:8-103 79
1 15:8-104 48
1 15:8-105 197
1 15:10-1 43
1 15:10-2 187
1 15:10-3 123
1 15:10-4 83
1 15:10-5 123
27 15:11-30 82
27 15:11-31 121
27 15:11-32 83
27 15:11-33 113
27 15:11-34 66

Raw Qubit readout (Google Sheet): 20160725_DNA_quant_Qubit_Ava_abalone_WS

Master spreadsheet for these, and future, samples for this project (Google Sheet): Ava WS Transmission DNA Extractions

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DNA Extraction & Quantification – Ava Withering Syndrome Transmission Study Tissues

Isolated DNA from 24 tissue samples provided by Ava. Presumably, the tissues were digestive gland and I believe they were preserved in ethanol. The list of samples can be seen in the results below.

DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s protocol with the following options:

  • Samples were briefly homogenized (due to their stiffness resulting from ethanol fixation) in the InhibitEX Buffer using disposable plastic pestles.
  • Homogenized tissue was incubated at 95C to maximize cell lysis
  • Followed “human DNA analysis” protocol for remainder of protocol (to maximize sample recovery)
  • Eluted DNA with 100μL Buffer ATE

After extraction, the samples were quantified using the Roberts Lab Qubit 3.0 (Life Technologies) using the Qubit ds DNA BR reagents. Used 1μL of each sample.

Samples were stored at 4C in FSH240 in the box “Ava WS Transmission DNA Extractions by Sam Box 1″. See the master spreadsheet at the bottom of this post for specific sample locations within this box.

Results:

cage_number accession_number concentration(ng/uL)
21 15:11-89 168
21 15:11-90 69.2
21 15:11-91 70.4
21 15:11-92 58.8
21 15:11-93 61.6
17 15:11-84 48
17 15:11-85 80
17 15:11-86 138
17 15:11-87 68
17 15:11-88 18.2
23 15:9-144 60
23 15:9-145 72
23 15:9-146 121
23 15:9-147 159
23 15:9-148 41.8
20 15:11-100 29
20 15:11-101 133
20 15:11-102 116
20 15:11-103 163
20 15:11-104 162
9 15:10-25 226
9 15:10-26 133
9 15:10-27 182
9 15:10-28 194

 

Raw Qubit readout (Google Sheet): 20160721_DNA_quant_Qubit_Ava_abalone_WS

Master spreadsheet for these, and future, samples for this project (Google Sheet): Ava WS Transmission DNA Extractions

 

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qPCR – Black Abalone with XC Prophage Portal Primers

Ran qPCR with black abalone samples from the 1st and 2nd experiments to see if the Xenocal prophage portal gene is detected.

Master mix calcs (Google Sheet): 20160421 – qPCR Black Abs XenoCal phage portal

All samples were run in duplicate.

Black abalone sample 08:13-2 was run as a positive control.

Plate layout, cycling params, etc. can be seen in the qPCR Report (see Results below).

Results:
qPCR Report (PDF): Sam_2016-04-21 14-11-09_CC009827.pdf
qPCR Data File (CFX): Sam_2016-04-21 14-11-09_CC009827.pcrd

Two samples failed to produce amplification: 06:6-44 and 07:12-18. All other samples amplified. Will compile this data with WSN and RLOv DNA helicase and send along to Carolyn and Stan.

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DNA Isolation – Black Abalone 2nd Experiment 08:13 Accessions

Isolated DNA from EtOH-preserved black abalone digestive gland tissue from the 2nd black abalone experiment.

There’s some odd background in regards to these samples which I previously described here that might be worth reviewing.

DNA was isolated using the QIAamp Fast DNA Stool Kit (Qiagen). Tissues were weighed and briefly homogenized with a disposable pestle in InhibitEX Buffer. Manufacturer’s protocol was followed. DNA  was eluted in 100μL of Buffer ATE and quantified on the Roberts Lab Qubit3.0 (ThermoFisher) using 1μL with the Qubit dsDNA Broad Range assay.

Results:

Google Sheet: 20160421_DNA_isolation_08:13_subset

 

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Data Aggregation – WS RLO and RLOv DNA Helicase qPCR and WS RLO Infection Intensities

Carolyn asked me to send her the data described above.

RLOv DNA helicase qPCR data were grabbed from the qPCR I ran on 20160106.

The qPCR data for withering syndrome RLO were culled from these three different spreadsheets:

 

The summary is below. I have emailed a copy of the spreadsheet to Carolyn.

Google Sheet: 20160404_Summary_RLO_RLOvDNAhelicase_qPCR_HistoIntensities

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qPCR – XenoCal Prophage Portal Primers on RLO/RLOv positive/negative samples

Stan Langevin and Carolyn wanted to see if this particular gene was found in the withering syndrome (RLO) or phage (RLOv) genomes. I previously identified 10 samples of each of the following combinations:

  • RLO-/RLOv-
  • RLO-/RLOv+
  • RLO+/RLOv-
  • RLO+/RLOv+

Master mix calcs are here (Google Sheet): 20160331 – qPCR Black Ab 08:13 XenoCal phage portal check

All samples were run in duplicate.

Plate layout, cycling params, etc can be viewed in the qPCR Report (see Results below).

Results:
qPCR Report (PDF): Sam_2016-03-31 08-51-11_CC009827.pdf
qPCR Data File (CFX): Sam_2016-03-31 08-51-11_CC009827.pcrd

The results are definitely interesting!

Quick summary: Amplification only seen in RLO+/RLOv- samples!

Oddly, there is no amplification in the other group of RLO+ samples (RLO+/RLOv+). Based on the fact that there is amplification in RLO+/RLOv- samples, which implies this prophage portal gene is present in withering syndrome, we would expect to also have amplification in the other group of samples that are positive for withering syndrome.

Compiled qPCR data with WSN1, RLOv_DNA_helicase, and XenoCal prophage portal gene (Google Sheet): 20160331_qPCR_summary_RLO_RLOv_pos_negs

 

Amplification Plots (PINK= RLO+/RLOv-, BLUE = RLO-/RLOv-; GREEN = RLO-/RLOv+; BLACK = RLO+/RLOv+)

 

Melt Curve Plots (see amplification plots for color scheme)

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Sample ID – RLO+/RLOv- Samples for XenoCal Prophage Portal qPCRs

Earlier today I identified a bunch of samples that will be used to test out the XenoCal prophage portal primers to see if it’s present in the withering syndrome bacteria (RLO) or in the wither syndrome phage (RLOv). However, my search showed that we didn’t have sufficient samples that were RLO+/RLOv-.

I contacted Carolyn and she provided me with a spreadsheet summarizing the 2nd black abalone withering syndrome challenge. This data has samples that are known to be RLO+ (via qPCR – data is in spreadsheet) and should be RLOv- (will have to verify via qPCR before proceeding).

Google Sheet: MAIN data summary- black abalone 2nd study Nov  19 2012 for stats with question re trial 2 slides

It looks like the 08:13 accessions are all RLO+. I will track down these samples and verify they are RLOv- before proceeding with the XenoCal prophage portal qPCRs.

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Sample ID- XenoCal Prophage Portal Tests

Now that the XenoCal prophage portal primers appear to be in working order, Carolyn wants me to test them out on 10 samples with the following status':

  • RLO-/RLOv-
  • RLO-/RLOv+
  • RLO+/RLOv-
  • RLO+/RLOv+

In order to quickly identify samples with these qualifications, I ran a SQL query on the following spreadsheet that contain qPCR data for both withering syndrome (RLO) and the phage (RLOv):

I saved the following worksheets from the above Google Sheet as CSV files:

  • water 2010
  • water 2011

These were imported to SQLite as I’ve previously done.

The two sheets were renamed for use in SQLite, respectively:

  • AbEndoWater2010
  • AbEndoWater2011

Here are the four queries I ran to obtain the four combinations of RLO/RLOv samples listed above

RLO-/RLOv-

sqlite> SELECT '2011_H2O', "DNA Tube Label", "Mean Cq", "RLOv_mean_Cq" FROM AbEndoWater2011 WHERE "Mean Cq"=0 AND "RLOv_mean_Cq"=0

 

RLO-/RLOv+

sqlite> SELECT '2011_H2O', "DNA Tube Label", "Mean Cq", "RLOv_mean_Cq" FROM AbEndoWater2011 WHERE "Mean Cq"=0 AND "RLOv_mean_Cq">0

 

RLO+/RLOv-

sqlite> SELECT '2011_H2O', "DNA Tube Label", "Mean Cq", "RLOv_mean_Cq" FROM AbEndoWater2011 WHERE "Mean Cq">0 AND "RLOv_mean_Cq"=0

 

RLO+/RLOv+

sqlite> SELECT '2011_H2O', "DNA Tube Label", "Mean Cq", "RLOv_mean_Cq" FROM AbEndoWater2011 WHERE "Mean Cq">0 AND "RLOv_mean_Cq">0

 

Results:

It looks like we do not currently have 10 samples that are RLO+/RLOv-. I will contact Carolyn to see if she happens to know of any samples that are RLO+, but do not contain (or, should not) any RLOv.

The full list of results can be seen in the Google Sheet below.

Google Sheet: 20160322_RLO_RLOv_pos_negs

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qPCR – Check New Withering Syndrome p18RK7 Plasmid Standard Curve Dilution

This is a qPCR to test the fresh p18RK7 dilution I made earlier today, and verify it works well (i.e. linear spread, good R^2 value, same Cqs as previous curve dilution, etc.).

Master mix calcs (Google Sheet): 20150316 – qPCR WS p18RK7 Curve Check

I made three separate master mixes to check the curve three times.

All samples were run in duplicate. Plate layout, cycling params, etc. are in the qPCR Report (see Results below).

Baseline threshold was manually set to 580, as previously determined by Lisa for this assay.

Results:

qPCR Report (PDF): Sam_2016-03-16 17-04-05_CC009827.pdf
qPCR Data File (CFX): Sam_2016-03-16 17-04-05_CC009827.pcrd

Overall, the curve looks good and has very comparable Cq values at each dilution of the curve. Will use this for future withering syndrome qPCR assays and will send an aliquote of each dilution to Colleen Burge.

MASTER MIX #1


 

MASTER MIX #2

 


MASTER MIX #3

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